PB84-223023
Causes of Papillosr.as on Pish Exposed to
Chlorinated Sewage Effluent
Auburn Univ., AL
Prepared for
Environmental Research Lab., Gulf Breeze, FL
Jul 84
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EPA-600/3-84-076
1984
CAUSES OF PAPILLOMAS ON FISH EXPOSED
TO CHLORINATED SEWAGE EFFLUENT
by
John M. Grizzle and Paul Melius
Department of Fisheries and Allied Aquacultures
and Department of Chemistry
Auburn. University, Alabama 36849
CR809336010
Project Officer
William Davis
Environmental Research Laboratory
Gulf Breeze, Florida 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
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TECHNICAL REPORT DATA
(Please read Instnuti^ni on the reverse before completing}
. REPORT NO.
EPA-6GO/3-84-076
3. RECIPIENT'S ACCESSION NO.
PS83 223023
4. TITLE AND SUBTITLE
CAUSES OF PAPILLOMAS ON FISH EXPOSED TO CHLORINATED
SEWAGE EFFLUENT
5. REPORT DATE
July 1934
6. PERFORMING ORGANIZATION CODE
7. AUTHORS)
J.M. Grizzle and Paul Melius
I. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
tO. PROGRAM ELEMENT NO.
Department of Fisheries and Allied Aquacultures &
Department of Chemistry
Auburn University, Alabama 36849
11. CONTRACT/GRANT NO.
CR80933010
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Environmental Research Laboratory
Office of Research & Development
Gulf Breeze, FL 32561
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/04
IS. SUPPLEMENTARY NOTES
16. ABSTRACT.
This research was initiated to determine the cause of oral papillomas in
black bullheads (Ictalurus melas) from the final oxidation pond of the
Tuskegee, Alabama, sewage treatment plant. The water in this pond was
chlorinated effluent from the sewage treatment plant.
The presence of a carcinogenic and mutagenic chemical in the effluent of a
sewage treatment plant was indicated by papillomas developing on caged black
bullheads, glucuronosyl transferase induction in caged channel catfish, and
Ames-test mutagenicity of water extract. Unlike previously studied fish
papillomas, virus-like particles were not present in the tumor cells. Although
mutagenic and carcinogenic chemicals have not been identified in the
wastewater, chlorine is implicated as a factor contriubting to the induction of
the papillomas because the prevalence of papillomas on wild black bullheads
exposed to the effluent decreased from 73% to. 23% after the dtlorination rate
was reduced.
This report was submitted in fulfillment of Grant No. LCR8Q9336010 by
Auburn University under the sponsorship of the U.S. Environmental Protection
Agency. This report covers the period from 12 October 1981 to 11 October 1983,
and work was completed as of 11 October 1983.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS-OPEN ENDED7SRMS C. COSATI Field/Group
18. DISTP.iSUTSON STATEMENT
Release to public
19. SECURITv_<-^r.35 /Viu Kep
unclassified
21. .N'Q. OF PAGES
33.
20. SECURITY CLASS , rtlii pJtt
22.
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EOITION >s
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
11
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FOREWORD
The protection of our estuarine and coastal areas from damage caused by
toxic organic pollutants requires that regulations restricting the introduction
of these compounds into the environment be formulated on a sound scientific
basis. Accurate information describing dose-response relationships for organisms
and ecosystems under varying -conditions is required. The EPA Environmental
Research Laboratory, Gulf Breeze, contributes to this information through
research programs aimed at determining:
the effects of toxic organic pollutants on individual species and
communities of organisms;
the effects of toxic organics on ecosystem processes and components;
the significance of chemical carcinogens in the estuarine and marine
environments.
Few studies have reported carcinogenic expressions in populations of
fishes, and still fewer researchers have been able to manipulate the frequency
of the tumor expression. In this report, a population of catfish exposed to
chlorinated sewage effluent are caged and compared with related species, and
efforts to separate the tumor inducer were undertaken. Results indicated that
the incidence of papillomas declined after the chlorination rate was reduced.
These data provide an intriguing link between waste water treatment and tumor
induction in fish.
Enos
Director
Environmental Research Laboratory
Gulf Breeze, Florida
TM
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ABSTRACT
This research aas initiated to determine the cause of oral papillomas in''
black bullheads (Ictalurus melas) from the final oxidation pond of the Tuakegee,
Alabama, sewage treatment plant. The water in this pond was chlorinated efflu-
ent from the sewage treatment plant.
The presence of a carcinogenic and mutagenic chemical .in the effluent of a
sewage treatment plant was indicated by papillomas developing on caged black
bullheads, glucuronosyltransferase induction in caged channel catfish, and
Ames-test mutagenicity or water extract. Unlike previously studied fish
papillomas, virua-like particles were not piresent in. the tumor cells. Although
mutagenic and carcinogenic chemicals have not been identified in the wastewater,
chlorine is implicated as a factor contributing to the induction of the
papillomas because the prevalence of papillomas on wild black bullheads exposed
to the effluent decreased from 73% to 23% after- the chlorination rate was
reduced. .
\ ./
This report was subsitted in fulfillment of Grant No. CR80933&010 by Auburn
University under the sponsorship of the U.S. Environmental Protection. Agency.
This report covers the period from 12 October 1981 to 11 October 1983, and work
was completed as of 11 October 1983.
IV
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CONTENTS
Page
Abstract iv
Figures . vi
Tables vii
Acknowledgments ....' viii
1. Introduction 1
2. Conclusions and Recommendations . 2
3. Study Site 3
4. Materials and Methods . 6
5. Results 10
6. Discussion 19
References 21
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FIGURES
Number Page
1 Schematic plan of Che Tuskegee, Alabama, sewage treatment plant ... 4
2 Total residual chlorine (monthly averages) in water leaving the
. chlorine contact chamber of the Tuskegee sewage treatment plant . . 5
3 Prevalence of papillotnas on wild black bullheads from the final
oxidation pond of the Tuskegee sewage treatment plant . . 11
4 Oral papillcina on the mouth fornix of a black bullhead. 12
5 Superficial portion of an oraL papilloma. Epithelial cells,
goblet cells, and alarm substance cells are arranged around
lumens forming cell nests . 13
6 Response of Salmonella typhimurium strain TA100 in the Ames test
to organic extracts of water collected at the inlet or outlet of
the final oxidation pond of the Tuskegee, Alabama, sewage treatment
plant 16
7 UDP-glucuronosyltransferase activity (nmol/min/ng protein)in
liver microsomes of caged channel catfish in the final oxidation
pond of the Tuskegee, Alabama, sewage treatment plant or in a
control pond 16
VI
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TABLES
Number
Maximum prevalence of oral lesions on caged fish in the final
oxidation pond of the Tuskegee sewage treatment plant ....
VII
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ACKNOWLEDGMENTS
The cooperation of the South Tuskegee Water Pollution Control Plant
personnel, is gratefully acknowledged. Auburn University staff and graduate
students that contributed to this project were Barrie Tan, Chalor Limsuwan,
Leonard Hochstedler, Paraskevi Karathanasis, Diane Biba, Richard Maxwell, Jai
Hosey, and Steve Horowitz.
Vlll
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SECTION 1
INTRODUCTION
A population of black bullheads in the final oxidation pond of the City of
Tuskegee, Alabama, sewage treatment plant had a 73% prevalence of oral
papillomas (Grizzle et al. 1981). These fish provided an opportunity to study
the cause of fish papillomas, especially as related to chlorinated sewage
effluent.
Sewage entering this treatment plant did not include discharge from
industries or institutions, but run-off water from residential and agricultural
areas entered the plant after rains. The process of chlorinating the sewage
after it passed through an aeration basin and a settling basin was considered a
possible source of carcinogens.
There has been only one previous report of a high prevalence of neoplasms
in aquatic animals living in chlorinated sewage effluent (Rose 1977). In that
case, a pond at Reese Air Force Base, Texas, contained tiger salamanders
(Ambystoma tigrinum) that had neoplastic and non-neoplastic lesions with a peak
frequency of 53%. Chemical carcinogens, especially perylene, were suspected of
causing the lesions.
Papillomas occur on many species of fish and have been associated with
pollution (Lucke and Schlumberger 1941; Russell and Kotin 1957; Cooper and
Keller 1969; Sonstegard 1977; Dawe and Harshbarger 1975) and tumorogenic viruses
(Edwards et al. 1977; Schwanz-Pfitzner 1976; McAllister et al. 1977). Other
fish tumors have a genetic origin (Siciliano et al. 1971; Vielkind et al. 1971).
Laboratory and field experiments were initiated to determine the cause of
the papillomas. Some of these experiments were a continuation of a previous EPA
cooperative agreement (CR807844010, Grizzle and Melius 1983). Objectives were
to: (1) determine the prevalence of papillomas on fish in the final oxidation
pond; (2) describe the histology and ultrastructure of the papillomas; (3)
determine if the papillomas would persist on black bullheads taken from the
final oxidation pond and placed in clean water; (4) chemically identify
mutagenic and carcinogenic chemicals in the water; (5) determine if the water
was mutagenic; (6) determine if tumors would develop in new fish placed intc the
final oxidation pond; (7) determine if hepatic-enzyme induction occurred in fish
exposure to the water (8)- determine if viruses were associated with the
papillomas by ultrastructural examination, transmission by cell-free homogenate,
and isolation of virus wLth cell-culture techniques; (9) determine if lesion?
would develop in brown bullheads exposed during embryonic development to a
concentrate of the final oxidation pond water; and (10) determine if tumors
would develop on black bullheads exposed to an organic concentrate of the final
oxidation pond sediment injected or applied topically to che mouth.
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SECTION 2
CONCLUSIONS AMD RECOMMENDATIONS
The Tuskegee, Alabama, sewage treatment plant final oxidation pond had
wild black bullheads with a 73% prevalence of oral papillomaa. Results of this
study indicated that the acidic organic, solvent extract of this oxidation pond
water was seasonally cucagenic to Salmonella typhimurium bacterial tester
strains TA98 and TA100 if activated with Arochlor-induced rat liver enzymes. No
evidence of a viral etiology for the papillonas was found during ultraatructural
studies of the lesions or during transmission attempts.
V
Caged black bullheads in the oxidation pond developed oral papillomae in
the saraa mouth location occupied by papilloraas in wild black bullheads. The
lesions in caged fish were probably related to the mutagenic chemicals in the
water because there was also induction of hepatic enzymes in fish exposed to the
final oxidation pond water. Neoplasms did not.develop in adult fiah or embryos
exposed to water or sediment extracts. '
^
The usefulness of exposing caged fish to water for detection of chemical
carcinogens was demonstrated by the development -of neoplasms on caged black
bullheads. The use of chemical analysis in this case failed to indicate that
rautagenic or carcinogenic chemicals were present. Laboratory exposures of black
bullheads and brown bullhead embryos to organic extracts of the Tuskegee
wastewater failed to cause tumors. Additional refinement of techniques may
improve the usefulness of laboratory exposures, but. presently field exposures .
of caged fish are more conclusive.
Chlorination was implicated in the induction of the papillomas because the
prevalence decreased after the chlorination rate was reduced. Further studies
should determine the identity of the rautagenic .and carcinogenic chemicals, the
role of chlorine in causing these tumors, and the persistence of the
carcinogenic chemicals after release into the receiving stream.
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SECTION 3
STUDY SITE
The 0.8-ha final oxidation pond of the Tuskegee, Alabama, sewage treatment
plant was designed for post-secondary treatment of sewage effluent. Water
retention time in the pond depends on the flow rate and averages 4-5 days.
Sewage treatment before the oxidation pond consists of sedimentation, activated
sludge secondary treatment, and disinfection by cholorination (Fig. 1). During
January-November 1979, monthly average residual chlorine concentration was
1.2-3.1 mg/liter in the effluent leaving the chlorine contact chamber and when
entering the final oxidation pond (Fig. 2). Since November 1979 this chlorine
concentration has ranged from 0.3 to 1.2 mg/liter. Residual chlorine
concentration of water leaving the final oxidation pond is usually.0.1 mg/liter,
approximately the same as before November 1979.
The- portion of Tuskegee's sewage that entered this plant contained only
domestic wastes; however, following heavy rainfall the flow rate into the plant
increased greatly, indicating that surface runoff from surrounding agricultural
and residential areas entered the sewage system.
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FIGURE 1. Schematic plan of the Tuskegee, Alabama, sewage treatmsat plant. A,
pumping station; B, grit removal; C, aeration basins; D, settling basins; E,
chlorine contact chamber; F, final oxidation .poad4
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DATE
FIGURE 2. Monr.aly averages for total residual chlorine (mg/liter) in water
leaving -the chlorine cotact of the Tuskegee sewage treatment plant.
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SECTION 4
MATERIALS AND METHODS
Fish were collected from the final oxidation pond of the Tuskegee, Alabama.
sewage treatment plant with gill nets and traps. During 2 bacterial epizootics,
moribund fish were picked up with a dip net. For determining prevalence of
papillomas, most papilloaaa were diagnosed grossly. Representative lesions were
examined by light microscopy to confirm the diagnosis. Fish were put back into
the pond if they were not necropsied or used in experiments. The papillomas on
26 adult black bullheads from the final oxidation pond were measured, the fish
were identified with tags, and then placed in 3-m diameter plastic pools at the
Alabama Agricultural Experiment Station, Auburn, Alabama, to determine the fate
of papillomas after the fish were placed in clean water.
.For light microscopy, tissues were fixed in 10% buffered formalin or
Bouin's, embedded in paraffin, sectioned at 6-inn thickness, and stained with
hematoxylin and eosin. Selected paraffin sections were also stained with
periodic acid-Schiff, Masson's trichrome stain, and Feulge^ reaction.
For transmission electron microscopy (TEM), 1-mnr' pieces of tissue fixed
in phosphate buffered glutaraldehyde and post-fixed in osmium tetroxide were
embedded in Eopn-Araldite, sectioned at approximately 70-raa thickness, and
stained with lead citrate and uranyl acetate.
The Alabama Pesticide Residue Laboratory, Auburn, Alabama, and the Alabama
Department of Public Health Laboratory, Montgomery, Alabama, used gas
chromatography to determine if toxic organic chemicals were in the water or
sediment of the final oxidation pond.
Extracts of the final oxidation pond water were tested for mutagenicity
with the Ames test (Ames et al. 1975) using Salmonella .typhmurium tester
strains TA 98 and TA 100 supplied by Dr. Anie!~IPositive controls were run with
benzo(a)pyrene. Three types of organic extracts of the pond water
for mutagenicity testing. For one procedure 10 liters water was filtered
through XAD resin and the organics were eluted with acetone. For the other
procedures, the pH of 2 liters of water was adjusted to either 1.0 with HC1
(for acid fraction) or 12-13 with NaOH (for basic fraction) and then extracted
three times with 200 ml of 25% ether and 75% hexane per liter of water. For all
procedures, the bulk of the solvents were removed by distillation. The last 20
ml of solvent was evaporated under a stream of nitrogen. Extracts were
dissolved in dimethylsulfoxide and filter sterilized before addition to the
agar. Concentration of the organic extract ranged from 0.3-3.0 mg/plate.
Black bullheads, brown bullheads (Ictalurus nebuloaus), yellow bullheads
(Ictalurus natalis), and channel catfish (IctalTTrus punctatus) were placed into
cagesInthefinal oxidation pond and a controlpond atthe Alabama Agricultural
Experiment Station. The 1-m cages were allowed to rest on the pond bottom
(sinking cages) or suspended off the bottom by floats (floating cages). Cages
were placed at three locations in the final oxidation pond: near the inlet
(inlet A), approximately 60 meters from the inlet (inlet B) or near the pond
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outlet (outlet). Most cages were stocked with 50 fish of a single species with
total lengths of the fish between 5-10 en. .
Glucuronosyltransferase (CDP-GT) and sulfotransferase (ST) were assayed in
livers of caged fish as part of a separate- EPA cooperative agreement to Dr. D.
R. Strength (Department of Animal and Dairy Sciences, Auburn University). .
Livers were homogenized in a Potter-Elvehjem-type homogenizer with teflon pestle
with 4 parts of 0.25 sucrose containing 0.01M Tris, pH 7.5. A clarified
hotnogenate was prepared by centrifuging at lO.OOOxg for 30 min. Microsomes were
sedimented at iOO.OCOxg for 1 hr, yielding the supernate and microsomal
fractions. The microsomes were washed twice in Tris-sucrose buffer, recovered
by centrifugation and suspended in 0.1M Tris buffer, pH 7.5 prior to use or
storage. Supernate fractions for immediate assay of ST were prepared by
homogenizing livers directly in 0.1M Tris buffer, pH 7.5, and centrifuging 1 hr
at lOO.OOOxg. Protein concentrations were determined by the procedure of Lowry
et al. (1951).
V
UDP-GT was assayed colorimetrically by the decrease in absorbance at 400 run
using p-nitrophenol as ar. acceptor. The reaction ixture contained the following
in umoles; p-nitrophenol, 0.12; UDP-glucuronic acid, 1.2; MgC^. 4; Tris
buffer, pH 7.5, 11; the total reaction volume of 0.2 ml also contained 40 ug of
Triton X-100 and 20 to 50 ug of microsomal protein. The reaction was started by
adding UDPGA and stopped after 10 min incubation at 37 C by adding 0.2 ml of
ethanol. The reaction mixture was then centrifuged for 10 min at l.OOOxg and
0.1 ml of the supernate was added to 0.9 ml of 0.1M EDTA, pH 9.6 and the
AAQO determined. The change in absorbance was determined from the
difference in A4QQ of identical reaction mixtures incubated zero and 10
min, respectively; the quantity of p-nitrophenol was ascertained from standard
solutions measured in the same medium.
t
ST activity was determined colorimetrically measuring the p-nitrophenol
released from PNPS in the presence of PAP. The reaction mixture from the
lOO.OOOxg supernate fraction of tissues in a total volume of 1.0 ml. The
reaction was conducted at 37 C for 10 min and was terminated by adding 2.5 ml of
ethanol. After centrifugation at lOOOxg for 10 min, the supernate was decanted
into a tube containing 0.5 ml of 0.2M glycine buffer, pH 10.4 and i nil of
distilled water (total volume used 5 ml). The absorbance at 400 rim was measured
in a spectrophotometer, or alternatively in a Klett-Summerson Colorimeter with
#42 filter. Zero time controls and standard solutions of p-nitrophenol were
measured in similar fixtures; the p-nitrohenol libertated from PNPS was ;
expressed in nmoles/min/mg protein.
Papillomas from final oxidation pond black bullheads were homogenized in
Hank's.balance salt solution (HBSS), filtered.through a 0.45-um filter, and .
injected subcutaneously into the mouth fornices of 19 adult black bullheads.'
Seven control black bullheads were injected with HBSS. Each fish was kept in a.
separate 40-liter aquarium and examined monthly.
Brown bullhead broodstock were obtained from Easterling's Fish Hatchery,
Clio, Alabama. On May 18, 1981, paired broodfish were placed in spawning pens
in an earthen pond at the Alabama Agricultural Experiment Station. Spawning
boxes were checked for eggs twice daily and egg-s were transferred to
recirulating aquaria. '
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Eggs wer» kept in a recirculating systems with 10, 15-liter aquaria.
Filtered pond water was used to fill the system initially aud to replace water
lost by leakage. Water drained from the aerated aquaria into a lower, 273-liter
reservoir and then flowed by gravity to the aquaria. Eggs ware incubated in a
recirculating system instead of the customary, flow-through troughs to avoid the
use of pond water with variable water quality.
Aqueous solutions of afltoxin Bj (AFB) were prepared from a 500 rag/liter
stock solution. To facilitate solubilization, 10 mg AFB (Sigma Co., St. Louis,
Mo.) were dissolved in 10 ml divnethylsulfoxide (DM50) and ethanol. Aflatoxin
Bi was not purified prior to use. DMSO was added/as a solvent after it was
observed that ethanol alone was insufficient for Solubilization. Aflatoxin Bj
concentrations of 0.5, 1.0 and 1.5 Qg/liter were made from the stock solution
with reconstituted soft water immediately before use.
Organic compounds were extracted from 15 liters of water collected at the
outlet from the final oxidation pond of the South Tuskegee Water Pollution
Control Plant, Macon County, Alabama, in mid-May 1981. The extraction
procedure, a modification of the technique used by Rappaport et al. (1979), used
a column of purified XAD-2 end XAD-7 resin. Unfiltered water flowed into the
column by gravity at 19 ml/miu. The column was dried under nitrogen to remove
residual water, and prganics were eluted with acetone. The acetone was then
dried under a nitrogen stream and the flask containing dried organics was stored
at -20 C.
Prior to embryo exposure, the yellowish-brown film of organics was
redissolved i.i 0.2-ml 95% ethanol and 0.4-ml DMSO, and diluted to 200 ml with
.reconstituted soft water. The final, 200-nil solution was a 75-fold -concentrata
of the effluent-water organics. Organic yield was not directly measured but
estimated to be between 0.7 mg/liter and 1.0 ing/liter (10 and 15 mg for 15
liters) based on later extractions of the same water.
When eggs.were five days of age, the egg mass was subdivided into groups of
approximately 200 eggs, and each group was placed in 300-ml glass dishes
containing 200 ml of the sublethal test solutions for one hour.
After hatching, fish were kept in aquaria until the yolk sac was absorbed.
Fish from the first replicate were transferred to 5700-liter plastic pools and
fish from the second replicate were kept by treatment group in cages in an
earthen pond on the Alabama Agricultural Experiment Station. Fry were fed on a
48% crude protein diet (Silver Cup No. 1), and after four months of age on a 36%
crude protein feed (Auburn Feed No. 3).
The only treatments sampled from the first replicate were the 0.5 mg/liter
AFB and solvent control, due to accidental drainage of the other plastic pools
after stocking. Samples of three to five fish from the two treatments surviving
in plastic pools were fixed in Bouin's fluid daily for two weeks after
fertilization, and at 25 days, three months, and six Lionths after hatching.
Samples of solvent control fish were fixed in Bouin's at these time periods for
histological examination of normal liver development and morphology. Eight
months after hatching, equal numbers of both the 0.5 mg/liter AFB and the
solvent control treatments were sampled as follows: 147 fixed in Bouin's, three
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dissected for fixation of livers for electron microscopy, and 20 were held for
tnicrosomal preparation of liver tissue.
All but two treatments of the second replicate, placed in cages, were lost
to bacterial infections during the induction period. Surviving treatments were
the 1.0 mg/liter AFB group and the Tuskegee effluent concentrate group. Brown
bullheads of the 1.0 mg/litcr AFB treatment were fixed in Bouin's at 25 days
(four) and at eight months (twelve) after hatching. Four brown bullheads
exposed to the Tuskegee effluent concentrate were fixed in Bouin's 25 days after
hatching. Remaining fish cf the effluent concentrate treatment were later lost
to bacterial infections.
Sediment from the inlet area was collected and allowed to dry for 96 hours.
The sediment was then further desiccated with Na2S04. Organics from the
sediment were then extracted using acetone and methylene chloride. The extract
obtained after distillation was dissolved in DMSO. Six fish were injected in
the right mouth fornix with 0.1 ml of the extract at a concentration of 83.3
mg/ml DMSO. Six control fish received an injection of 0.1 ral DMSO. Fish were
injected once every 2 months for a period of 9 months. The same extract was
also dissolved with DMSO to give a concentration of 50 ng/ral DMSO. The mixture
was-applied to the entire oral area of 8 black bullheads using a cotton swab.
Each fish was swabbed with a circular motion for 1 minute, at weekly intervale,
for a period of 6 months. Eight other fish that served as controls received
weekly, 1-minute swabs of DMSO only.
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SECTION 5
RESULTS
PAPILLOMAS ON WILD BLACK BULLHEADS IN THE FINAL OXIDATION POND
The prevalence of papillomas in wild, adult black bullheads in the final
oxidation pond decreased from 73% in 1980 to 23% in March 1983 (Fig. 3).
Another change in the papillomas was the size; the large papillomas seen
protruding from the mouth of black bullheads in 1979-1980 (Grizzle et al. 1981)
were not found in recent samples. All neoplasms were located in the oral cavity
or on the lips except for 1 papilloma and"*! dermal fibroma on the dorsal surface
of the heads of 2 specimens.
The only other species of fish present in the final oxidation pond before
May 1980 were green sunfish (Lepomis cyanellus) and golden shiners (Notemigonus
crysoleucas). During May and July 1980, 2,000 silver carp (Hypophthalmichtihys
molitrix) were released in the final oxidation pond. No tumors were found on
any of these species. s
The papillomas consisted of hyperplastic nucosa covering papillae of
submucosa (Fig. 4). Goblet cells and alarm substance cells were absent from
parts of the lesion; in other areas these iells were numerous and formed cell
nests (Fig. 5). Cytoplasmic inclusion bodies in cells near the tumor surface
were eosinophilic, PAS positive, and weakly Feulgen positive. The number of
mitotic figures was similar to that in normal oral mucosa.
Ultrastructurally, the oral papillomas of wild black bullheads from the
final oxiaation pond had epithelial cells resembling normal oral mucosa cells
and a central connective tissue column that was continuous wit'n the submucosa.
Near the tumor surface there were mucous cells, and plasma membranes of the
epithelial cells adhered closely with those of-adjacent cells. Desmosomes
occurred less frequently between the cells than in normal mucosa. Mitochondria,
endoplasimic reticulum, and ribosorces wee concentrated in the peripheral
cytoplasm of the epithlial cells. These surface cells often contained glycogan
particles among !.he tonofilamer.ts. The mid to near basal portion of the
papilloma consisted of stellate epithelial cells having more tonofilaments,
fewer organelles in the cytoplasm, and more intercellular space than epithelial
cells near the surface. Desmosomes occurred more frequently between the cells
in the basal area than near the. surface. Alarm substances occurred in the mid
portion of the epithelium and were surrounded by stellate cells. Numerous
lymphocytes were present in the basal region of-the tumor. The basal epithelial
cells contained numerous mitochondria, endoplasmic'reticulum, and ribosomes in
the cytoplasm. Plasma membranes adhered to the adjacent cells by desmosomes.
Intercellular spaces occurred less frequently in the basal region than in the
middle\portion of the tumor. * «
10
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0
Jan 80 Jul 80 Jan 81 Jul 81 Jan 82 Jut 82 Jan 83 Jul 83
DATE
FIGURE 3. Prevalence of papillomas on wild black bullheads from the final
oxidation pond of the Tuskegee sewage treatment plant. ;
-11
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4
J
FIGUPvE 4. Oral papillona on the mouth fornix of a black bullhead. X20.
12
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Reproduced From
best available copy.
FIGURE 5. Superficial portion of an oral ^pillcras- Epithelial cells, goblet
cells, and alarm substance cells are arranged around lumens forming cell nests
X60.
13
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ANALYTICAL CHEMISTRY OF FIRA^ OXIDATION POND WATER
Water collected on 16 November, 9 March 1980, 22 January 1982, and 3 March
1982 had unidentified nitrogen and phosphorus organic residues in the 100
ng/liter range. No significant residues of pesticides, herbicides, or PCB were
detected in the water or sediment. Water collected on 1 May 1981 contained
chloroform (9.0-13.5 ug/liter) and bromodichloromethane (0.7 ug/liter), but
chlorodiibromcraethane and bronoform were not found with a detection limit of
1.0 ug/liter. Chloroform concentrations were similar before chlorination and ia
the final oxidation pond.
MUTAGENICITY OF THE FINAL OXIDATION POND WATER
The acid fraction and the XAD extract were mutagenic to Salmonella
typhimurium tester strains TA98 and TA100 only if S-9 (aroclor-induced rat liver
enzymes) was present. The pmtagenicity of the organic extracts varied
seasonally (Fig. 6). The basic fraction was cot mutagenic. No differences have
been found between, extracts prepared by-organic extraction of acidified water
and extracts prepared by filtering the water through a resin column. Water
samples collected at both the inlet and outlet of the final oxidation pond were
mutagenic, indicating that the mutagenicity is not lost during retention in the
pond.
EXPOSURE OF FISH TO WATER IN THE FINAL OXIDATION POND
Papillomas developed on 5 of the black bullheads kept in cages in the final
oxidation pond (Table 1). The papillomas were located in the same area of the
mouth and had the same gross and histological appearance as the papillomas on
the wild black bullheads in the pond. Four of the papillomas developed between
467-537 days of exposure on fish -in a sinking cage near the outlet. One
papilloma developed in less than 57 days on a fish in a sinking cage at the
inlet B location. The small namber of fish developing papillomas prevents
conclusions concerning the importance of cage location and contact with the
sediment.
The most common types of lesions developing on fish confined to cages in
the final oxidation pond were oral mucosa hyperplasia and stomatitis usually
located in the mouth fornices. The incidence of oral lesions on caged fish in
the final oxidation pond varied depending on the fish species and the year that
the fish were exposed (Table 1). The highest incidence was in black bullheads
stocked during October 1980 and the incidence was less in each successive
stocking. Black bullheads had the highest prevalence of oral lesions of the A
species tested.. Similar oral lesions occurred on control black bullheads
(Table 1) but at a prevalence of 9% or less. There were no difference in
prevalence of hyperplasr.ir lesions between floating and sinking cages or between
inlet and outlet locations.
-------
Table 1. Maximum prevalence of oral lesions on caged fish in the final
oxidation pond of the Tuskegee sewage treatment plant. Control is the
maximum prevalence of hyperplastic oral lesions in caged fish kept in
a control pond. Neoplasms did not occur in control fish.
Species Stocking Days of Hyperplasia Papillomas Control
Date Exposure
% N % N days %
black bullhead
21
23
29
9
8
Oct
Feb
Oct
Apr
Jul
80
81
81
82
82
168
43
407
245
57
95
50
33
25
17
22
12
6
8
12
80
0
0
0
8
5
12
537
57
4
0
9
yellow bullhead 12 May 81 683 0 0 14
brown bullhead 29 Oct 81 228 0 0 0
channel catfish 16 Jan 81 436 30 10 0 ' 8
29 Oct 81 513 4 28 0 4
8 Jul 82 254 0 0 5
15
-------
400
3OO
02OO
"o
u.
v
|
z
100
o-o-o Inlet 1982
»-e-« Outlet 1981
o Outlet 1982
o o Intel 1983
O XAD -2
M
M J J A
Months
* -9
S O
FIGURE 6. Response of Salmonella typhi.muri.um strain TA100 in the Ames test to
organic extracts of water collected at the inlet .or outlet of the final
oxidation pond of the Tuskegee, Alabama, sewage treatment plant. The number of
colonies is the difference between test and control plates. The extracts tested
were prepared by solvent extraction of acidified water samples or by XAD-2 resin
absorption.
16
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INDUCTION OF HEPATIC ENZYME SYSTEMS
UDP-GI in channel catfish ire the final oxidation pond was higher than in
control channel catfish (Fig. 7). UDP-GT activity in other ictalurid species
and ST activity in all species tested in the final oxidation pond were not
consistently different than controls.
VIROIOGY OF THE PAPILLOMAS
Examination of papillomaa with TEM failed to reveal virus-like structures.
Fish injected with homogenised papillomas were observed 11-14 months without
signs of papilloaas or hyerplastic lesions.
EXPOSURE OF BROWN BULLHEAD EMBROYS TO FINAL OXIDATION POND WATER AND AFB
Trunk kidneys of fish, 25 days after exposure to 1.0 mg/liter AFB, had
atrophied glomeruli and renal tublar necrosis. Focal areas of atrophied
epithelial cells of renal tubules were dispersed among tubules of normal
structure. Livers of fish with- renal lesions appeared normal.
Twelve fish from the embryo exposure to 1.0 mg/liter AFB were examined
after eight months. Pathological effects were absent in all liver and kidney
tissue examined.
Exposure of brown bullhead embryos to 0.5 mg/liter AFB did not cause
pathological effects to livers and trunk kidneys at any development stage
examined. Liver tissue from a subsample of this group showed normal
ultrastructure, with individual variation in glycogen content.
No pathological effects were observed in livers or trunk kidneys of brown
bullheads 25 days after embryo exposure to an organic concentrate of Tuskegee
effluent water.
LABORATORY EXPOSURES OF BLACK BULLHEADS TO SEDIMENT EXTRACTS
Black bullheads injected with 0.1 ml DMSO developed necrosis at the
injection site. Bacterial infections occurred in the black bullheads being
swabbed with sediment extract. No tumors developed in either the injected-or
swabbed fish after 8. months.
17
-------
12
9
8
Q,
4
3
2
1
-E--
-C
- exposed
- control
Dec 81 Feb82 Apr 82 Jun82
6AfE
Nov82
FIG 7. UDP-glucuronosyltransferase (UDP-GT) activity (nmol/min/mg protein) in
liver Dvicrosoraes of caged channel catfish in the final" oxidation poad cf the
Tuskegee, Alabama, sewage treatment plant or in a control pond.
18
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SECTION 6
DISCUSSION
Histologically, the black bullhead papillomas were similar to those of
brown bullheads Ictalurus nebulosus described by Lucke and Schlumberger (1941),
Harshbarger (1977) considered bullhead papillomas to be a common type of fish
neoplasm. However the papillomas in the present case are distinctive because
most were uniformly located in the mouth fornices rather than in variable
locations.
The neoplastic nature of the papillomas we described is indicated not only
by the histopathology but also by their persistence after removal of the fish
from the final oxidation pond. If these lesions were inflammatory responses
they would have regressed more quickly.
The reason- for the uniform location of the papillomas in this population of
black bullheads may be related to the presence of the oral valve in the anterior
buccal cavity. The oral valve is a thin fold of mucosa located immediately
posterior to the upper and lower lips. The folds forming the oral valve
terminate laterally near the mouth fornix, and the lateral ends of these folds
were often inflammed in fish, including controls, kept in cages during this
study. The high incidence of mucosal hyperplasia in the oral valve area also
indicates that this location is sensitive to toxicants.
No evidence was found that a virus was associated with the, papiliomas of
black bullheads. This is in contrast to other fish papillomas in which viruses
can be seen in electron micrographs (Edwards et al. 1977) or isolated in cell
culture (Schw&nz-Pfitzner 1976). If a virus is associated with the black
bullhead papillo-nas, they are unlike those .previously reported to be present in
fish .papillomas.
In contrast to the direct-acting mutagens pre'sent in the neutral or basic
fraction of wastewater from other sewage treatment plants (Saxena and Schwartz
1979, Rappaport. et al. 1979, Gumming et al. 1980), the aciJic fraction of the
Tuskegee sewage 'effluent was most uutagenic but only after metabolic activation
by S-9. This indicates that the primary mutagen in the Tuskagee wastewater is
different than in previously reported cases of mutagenic wastewater. Both the
activated sludge process (Rappaport et al. 1979) and disinfection with chlorine
(Cunnaing et al. 1980) have been implicated in increasing mutagenicity; both of
these factors could be involved .in the present case.
The cause of seasonal changes in mutagenicity of the Tuskegee wastewater is
unknown but may be related to higher temperatures during chlorination because
the pe?k mutagenicity is during summer. Seasonal variation in wastewater
contaminants such as pesticides could also be involved. Accumulation or organic
material in filter beds of treatment plants was suggested as a cause of seasonal
variation in mutagenicity of drinking water (Grimm-Kibalo 1981)". Variation in
domestic wastewater mutagenicity could be related to variation in drinking water
mutagenicity. . . ;,
19 .
-------
The induction of UDP-GT in channel catfish confined to the final oxidation
pond indicates exposure to a toxicnt. Similar induction occurs in rats and fish
given oral or intraperitoneal doses of carcinogens. The seasonal ch&nges in
UDF-GT activity also strongly indicates the effect of temperature on enzyme
induction in fish. The conjugase-enzyme induction in channel catfish but not in
black-bullheads may be related to the greater susceptibility of black bullheads
to the tumor inducing effects of chemical carcinogens in the final oxidaiton
pond.
The usefulness of exposing caged fish to water for detection of chemical
carcinogens was demonstrated by the development of neoplasms on caged black
bullheads. The use of chemical analysis in this case failed to indicate that
mutagenic or carcinogenic chemicals were present. Laboratory exposures of black
bullheads and brown billhead embryos to organic extracts of the Tuskegee
wastewater failed to cause tumors. Additional refinement of techniques may
improve the usefulness of laboratory exposures, but presently field exposures
of caged fish are more conclusive.
The role of chlorine in causing the black bullhead papillomas was indicated
by the reduced prevalence of tumore after the chlorination rate was reduced.
Additional study of the Tuskegee, Alabama, sewage treatment plant is warranted
to determine the identity of the carcinogenic chemicals and the relationship
between chlorination and tumors.
20
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